摘要
目的探究长链非编码核糖核酸生长组织特异性转录体5(LncRNA GAS5)通过AKT/mTOR通路对结肠癌SW480细胞增殖和凋亡的影响。方法体外培养人结肠癌细胞SW480,随机分为对照组、LncRNA GAS5过表达质粒组、LncRNA GAS5空载质粒组、LncRNA GAS5 siRNA组、LncRNA GAS5 siRNA阴性对照组,分别转染质粒及siRNA后,采用实时荧光定量(q RT-PCR)检测各组细胞LncRNA GAS5表达,采用CKK-8、细胞克隆形成实验检测细胞增殖情况,采用流式细胞术检测细胞凋亡情况,免疫印迹检测各组细胞凋亡相关蛋白[B淋巴细胞瘤-1(Bcl-2)、关联BCL-2的蛋白质X(Bax)、半胱氨酸蛋白酶-3(caspase-3)]、AKT/mTOR通路蛋白[丝氨酸/苏氨酸蛋白激酶(AKT)、磷酸化丝氨酸/苏氨酸蛋白激酶(p-AKT)、哺乳动物雷帕霉素靶点(mTOR)、磷酸化哺乳动物雷帕霉素靶点(p-mTOR)]表达情况。结果与对照组相比,LncRNA GAS5 siRNA组SW480细胞LncRNA GAS5表达、凋亡率、Bax及caspase-3蛋白表达降低,细胞活力、相对细胞克隆形成、Bcl-2蛋白表达、p-AKT/AKT、p-mTOR/mTOR升高,差异均有统计学意义(P<0.05);LncRNA GAS5过表达质粒组SW480细胞LncRNA GAS5表达、凋亡率、Bax及caspase-3蛋白表达升高,细胞活力、相对细胞克隆形成、Bcl-2蛋白表达、p-AKT/AKT、p-mTOR/mTOR降低,差异均有统计学意义(P<0.05);LncRNA GAS5 siRNA阴性对照组、LncRNA GAS5空载质粒组SW480细胞各指标差异无统计学意义(P>0.05)。结论上调LncRNA GAS5可抑制SW480细胞增殖,促进其凋亡,可能是通过抑制AKT/mTOR通路激活实现的。
Objective To investigate the effects of long noncoding RNA-growth arrest-specific transcript 5(LncRNA GAS5)on proliferation and apoptosis of colon cancer SW480 cells via AKT/mTOR pathway. Methods Human colon cancer SW480 cells were cultured in vitro and randomly divided into control group,LncRNA GAS5 over expression plasmid group,LncRNA GAS5 empty vector group,LncRNA GAS5 siRNA group and LncRNA GAS5 negative control group. After transfection of plasmid and siRNA respectively,the expression of LncRNA GAS5 was detected by real-time fluorescence quantitative analysis(qRT-PCR);the CKK-8 and cell clone formation assay were used to detect cell proliferation;flow cytometry was used to detect cell apoptosis,and the expressions of apoptosis-related proteins[B-cell lymphoma-2(Bcl-2),BCL-2-associated X protein(Bax),Cysteinyl aspartate-specific proteinase-3(caspase-3)],AKT/mTOR pathway proteins[protein Kinase B(AKT),phosphorylated protein Kinase B(p-AKT),mammalian target of rapamycin(m TOR),phosphorylated mammalian target of rapamycin(p-mTOR)]were detected by Western blot. Results Compared with the control group,the expression of LncRNA GAS5,apoptotic rate,expressions of Bax and caspase-3 in SW480 cells of LncRNA GAS5 siRNA group decreased,while the cell viability,relative cell clone formation,Bcl-2 protein expression,pAKT/AKT,p-mTOR/mTOR increased,and the differences were significant(P<0.05). The expression of LncRNA GAS5,apoptotic rate,expressions of Bax and caspase-3 in SW480 cells of LncRNA GAS5 over expressed plasmid group increased,while the cell viability,relative cell clone formation,Bcl-2 protein expression,p-AKT/AKT,p-mTOR/mTOR decreased,and the differences were significant(P<0.05). There was no significant difference in the parameters of SW480 cells between LncRNA GAS5 siRNA negative control group and LncRNA GAS5 empty plasmid group(P>0.05). Conclusion Up-regulation of LncRNA GAS5 could inhibit the proliferation and promote apoptosis of SW480 cells,probably by inhibiting the activation of AKT/mTOR pathway.
作者
蔡国强
陈雪军
王峰
CAI Guo-qiang;CHEN Xue-jun;WANG Feng(The First People′s Hospital of Kunshan City,Kunshan,Jiangsu 215300;Department of Oncology,Affiliated Hospital of Nantong University,Nantong,Jiangsu 226019,China)
出处
《热带医学杂志》
CAS
2020年第2期185-190,共6页
Journal of Tropical Medicine
基金
江苏省卫生计生委2016年度面上科研课题(H201620)。
